Abstract
Pyruvate dehydrogenase, isocitrate dehydrogenase, and a-ketoglutarate dehydrogenase of the mitochondrial matrix have been shown to have increased activities in the presence of Ca2+ ions in vitro (Denton and McCormack, 1985; Hansford, 1985). However, these studies were conducted modulating isolated enzyme activity, and show increases in substrate affinity, but not universally in maximum velocity. Furthermore, the effects of these enzyme activities on overall mitochondrial metabolic pathway flux has not been determined. We thus propose to verify if oxidative phosphorylation supported by different substrates (pyruvate, isocitrate, malate, a-ketoglutarate, glutamate, palmitoyl-CoA and glycerol phosphate) is modulated by Ca2+ in intact isolated mitochondria. This will be tested in isolated liver mitochondria, since a rich metabolic regulation by Ca2+ occurs in this tissue. Furthermore, liver mitochondrial Ca2+ homeostasis is altered by diet (Menezes-Filho et al., 2017; Menezes-Filho et al., 2019). Extramitochondrial Ca2+ will be added at different concentrations, with or without mitochondrial calcium uniporter (MCU) inhibition by ruthenium red, to modulate both extra and intramitochondrial Ca2+. Matrix Ca2+ will also be removed by incubation with BAPTA AM. The effects on oxygen consumption, ADP/O ratios and calibrated inner mitochondrial membrane potential measurements (Kowaltowski et al., 2002) will be monitored. These experiments will determine if extra or intramitochondrial Ca2+ modulate oxidative phosphorylation supported by different substrates, which surprisingly has not been verified to date. Respiratory rates will also be determined by Seahorse Extracellular Flux analysis (Cerqueira et al., 2016; Chausse et al., 2019) in hepatocyte cell lines in the presence and absence of MCU and NCLX activities, to determine the role of mitochondrial Ca2+ transport on oxidative phosphorylation in vivo. Cells incubated with BAPTA-AM, thapsigargin or ionomycin will be used as controls for low and high intracellular Ca2+. In addition to possibly regulating specific mitochondrial metabolic pathway activities, mitochondrial Ca2+ uptake changes cytosolic Ca2+ dynamics, and therefore may also influence cytosolic metabolic pathways. We will thus investigate the influence of mitochondrial Ca2+ transport on global cellular metabolic activity, by verifying the effects on MCU or NCLX inhibition on cell metabolism. Inhibitors of ER SERCA and store operated Ca2+ entry (Rahman and Rahman, 2017), as well as intracellular Ca2+ chelation with BAPTA-AM, will be used for comparison of the effects of other Ca2+ transport systems. The relative oxidation rates of lipids, glucose and glutamine will be determined by Seahorse Extracellular Flux analysis in the presence of these substrates and inhibitors of the metabolic pathways (Pike Winer and Wu, 2014). Results will be corroborated by measurements of phosphorylation levels of key metabolic regulatory enzymes (including phosphofructokinase/fructose biphosphatase 1 and 2, acetyl CoA carboxylase, pyruvate kinase, glycogen phosphorylase, glycogen phosphorylase kinase) and measurements of NA(P)D+/NAD(P)H in mitochondria and the cytosol. Lactate, glutamate and major lipid species will also be quantified. This will be conducted in collaboration with Prof. Sayuri Miyamoto, already an established collaborator in our metabolic studies (Menezes-Filho et al., 2017; Chausse et al., 2019). Cerqueira et al. 2016 FEBS J 283:822-833.Chausse et al., 2019 Biosci Rep 39: BSR20190072.Kowaltowski et al 2002 J Biol Chem 277:42802-42807.Menezes-Filho et al 2017 Free Radic Biol Med. 110:219-227.Menezes-Filho et al 2019 Biochim Biophys Acta Bioenerg 1860:129-135.Pike Winer and Wu, 2014 PLoS One 9:e109916.Rahman and Rahman 2017 Sci Rep 7:12881. (AU)
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